Human immunodeficiency virus (HIV) is an RNA virus that is the cause of acquired immunodeficiency syndrome (AIDS). This devastating disease is estimated to have killed 20 million people to date, and approximately 36 million people worldwide are HIV positive. Of the thirteen viral encoded proteins, two (reverse transcriptase and protease) have been targeted by drug inhibitors that can keep the pathology caused by the virus in check. However, the high mutation rate of the virus results in resistance to these drugs. Since most viruses are obligate intracellular parasites, new drug targets to consider are cellular oroteins that are required for completion of the virus life cycle. Our long-term goal is to characterize these oroteins and their role in the HIV-1 life cycle. This proposal is aimed at developing a genetic approach to dentify these cellular proteins. To this end we propose two specific aims. 1. Isolate genetic mutants in human cells that are resistant to infection by HIV. We will multiply infect a highly mutagenized population of HeLa cells with an HIV-1 based vector that transduces a toxic gene. Expression of this gene leads to cell death. We will isolate and expand HeLa clones that survive this selection, and confirm the resistance phenotype. We will establish that the block is specific for infection, and identify the point in the viral life cycle where the block is imposed. 2. Rescue the resistant phenotype of the clones by complementation cloning. We will use a novel high throughput semi-automated approach to rescue the resistance phenotype. 17,000 unique full length cDNAs in expression vectors are arrayed in single well formats in 384 well plates. Hence the identity and position of each clone is known. These cDNA clones will be transfected into the mutant clones isolated in specific aim 1 in single well format and these transfectants will be transduced with an HIV-1 vector that codes for luciferase, cDNA clones that show a significant increase (over the average of all 17,000 datapoints) in luciferase activity will be identified and expanded. We will confirm (i) the ability of the cDNA clone to rescue the resistant phenotype, (ii) that rescue is specific for infection and (iii) that it uniquely rescues specific Hela clones.